In crystallo observation of active site dynamics and transient metal ion binding within DNA polymerases

DNA polymerases are the enzymatic catalysts that synthesize DNA during DNA replication and repair. Kinetic studies and x-ray crystallography have uncovered the overall kinetic pathway and led to a two-metal-ion dependent catalytic mechanism. Diffusion-based time-resolved crystallography has permitted the visualization of the catalytic reaction at atomic resolution and made it possible to capture transient events and metal ion binding that have eluded static polymerase structures. This review discusses past static structures and recent time-resolved structures that emphasize the crucial importance of primer alignment and different metal ions binding during catalysis and substrate discrimination.

DNA polymerases are essential enzymatic catalysts that synthesize DNA. Based on their sequence homology, DNA polymerases are divided into A, B, C, D, X, Y, reverse transcriptase (RT), and primase and polymerase (PrimPol) families. As a diverse group of enzymes, they function during different stages of DNA replication and repair, prefer different DNA substrates, and vary in subunit compositions, catalytic rate, fidelity, and processivity Yang and Gao, 2018). Despite the diversity in their various biological roles and biochemical properties, early crystal structures revealed that DNA polymerases resemble a right-hand shape with the active site residing in the palm domain, nucleotide binding site at the finger domain, and the DNA binding site at the thumb domain [ Fig. 1(a)] (Steitz, 1999). The finger domain in replicative DNA polymerases undergoes an open-to-close conformational change upon correct nucleotide binding, whereas some polymerases specialized in translesion DNA synthesis do not exhibit finger domain movement (Johnson, 1993;Steitz, 1999;Yang, 2014). DNA synthesis requires divalent metal ions as cofactors (Yang, 2014). Two-metal ions (Me 2þ ) are coordinated by conserved negatively charged residues within the polymerase active site [Fig. 1(b)]. The A site metal ion (Me 2þ A ) binds near the primer terminus of the DNA duplex, while the B site metal ion (Me 2þ B ) is coordinated by the incoming nucleotide phosphate oxygens. Much efforts in capturing static crystal structures of polymerases complexed with various DNA substrates, drug compounds, and damaged DNA lesions have allowed enzymologists to further understand the function of polymerases during DNA replication and repair Yang and Gao, 2018). The substantial plasticity of the active site when polymerases interact with different DNA templates, nucleotide substrates, and metal ions suggests the existence of rich dynamic events and their critical roles during DNA synthesis.
Kinetic methods have elucidated the overall catalytic process of DNA synthesis and suggested that most polymerases catalyze DNA synthesis through similar kinetic pathways (Johnson, 1993;Joyce and Benkovic, 2004;Raper et al., 2018;Wu et al., 2017;Yang and Gao, 2018). First, DNA and the incoming nucleotide bind subsequently to the polymerase active site. Afterward, the 3 0 -OH of the primer becomes deprotonated and attacks the a-phosphate of the incoming nucleotide, followed by sequential release of the Me 2þ and pyrophosphate. At last, the release or translocation of DNA prepares the polymerase for the next round of DNA synthesis. Pre-steady state kinetics investigating nucleotide incorporation and misincorporation revealed that an anomalous conformational step right before and after nucleophilic attack may be the rate-limiting step in DNA synthesis and can contribute to polymerase fidelity Wong et al., 1991).
Early studies attributed this rate-limiting step to the open-to-close conformational change of the finger domain; however, the finger movement has been found to be excessively fast to be considered ratelimiting (Dunlap and Tsai, 2002;Rothwell et al., 2005;Tsai and Johnson, 2006). Some experiments further suggested that a "micro" closing conformational movement is rate-limiting, while others argue that no such conformational changes are needed to explain the observed kinetic data in substrate discrimination (Johnson, 2010;Oertell et al., 2016;Raper et al., 2018;Raper and Suo, 2016;Showalter and Tsai, 2002;Tsai and Johnson, 2006;Vande Berg et al., 2001). Direct visualization of the polymerase catalysis at atomic resolution is, thus, critical to illustrate the dynamic process of DNA synthesis and to clarify the role of the conformational checkpoint for catalysis and incorrect substrate discrimination.
Diffusion-based time-resolved crystallography has made it possible to observe transient events that occur during DNA synthesis in crystallo at atomic resolution (Chim et al., 2021;Freudenthal et al., 2013;Gao and Yang, 2016;Gregory et al., 2021;Nakamura et al., 2012;Samara and Yang, 2018;Vyas et al., 2015). Initial in crystallo studies observed a growing DNA chain after DNA translocation at high resolution by soaking A-family polymerase I Klenow fragment crystals in nucleotide-containing buffer (Johnson et al., 2003). However, rapid events immediately before and after the nucleophilic attack were not captured due to the slow diffusion rate of the nucleotide. Recently, an in crystallo reaction method that involves soaking DNA-polymerase-nucleotide (ternary) complex crystals in Me 2þ -rich buffer was developed (Chang et al., 2022;Gao and Yang, 2016;Gregory et al., 2021;Nakamura et al., 2012;Samara et al., 2017;Yang et al., 2017). The DNA synthesis reaction in polymerase ternary complex crystals with inhibitory metal ion Ca 2þ is initiated by the diffusion and binding of Mg 2þ and then halted by flash-quenching with liquid nitrogen freezing [ Fig. 1(c)]. Due to the small size and the fast diffusion rate of metal ions, Mg 2þ -diffusion-based in crystallo studies on Xfamily Pols b, k, and l and Y-family Pol g have revealed transient mechanistic events immediately preceding the nucleophilic attack as well as during and after the reaction. The dynamic mechanistic events, A and Me 2þ B are colored in green. The palm, finger, thumb, and exonuclease domains are colored in red, blue, green, and pink, respectively. (b) Schematic of the two-metal-dependent catalytic mechanism. The general base is denoted as B. The labeled residues are based on structures of Pol g. c, Metal ion soaking setup for in crystallo observation of DNA synthesis. The crystal of a ternary polymerase complex is grown with Ca 2þ in the hanging drop. The polymerase crystal is looped and first soaked in the equilibration buffer (crystallization condition with a pH of 7.0) to change the pH to pH 7.0. Afterward, the in crystallo reaction is initiated by looping and soaking the crystal in the reaction buffer (equilibration buffer that includes Mg 2þ or Mn 2þ ). After soaking for a brief period of time, the crystal is looped and soaked in the cryoprotection buffer (reaction buffer that includes 20% glycerol) for 1 s. Then, the crystal is rapidly immersed in liquid nitrogen and stored for x-ray analysis.
including primer terminus alignment, sugar pucker conformational changes, and transient metal ion binding, were found to play critical roles in DNA synthesis, repair, and fidelity control. Here, we reviewed recent static and dynamic structures of X-and Y-DNA polymerases with an emphasis on the power of time-resolved x-ray crystallography.

DYNAMICS OF PRIMER TERMINUS ALIGNMENT
During DNA synthesis, the 3 0 -OH terminal group of the primer needs to align properly with respect to the Me 2þ and the incoming nucleotide to undergo deprotonation and nucleophilic attack. Computational simulation studies have revealed that the primer has to be within 2.3-2.8 Å from the a-phosphate for efficient nucleophilic attack (Venkatramani and Radhakrishnan, 2010). A subtle movement or rotation in the primer by only 1-2 Å can place the primer 3 0 -O beyond this range and restrict chemistry. Moreover, the general base must approach the 3 0 -OH of the primer to carry out proton transfer before or concurrently during the nucleophilic attack. In order to capture ternary polymerase complexes, most early structural studies utilized DNA in which the primer strand contained dideoxynucleosides at the termini (Doubli e et al., 1998;Sawaya et al., 1994). Without a functional 3 0 -OH group, these structures found that the Me 2þ A was either missing or not properly aligned (Johnson and Beese, 2004;Vaisman et al., 2005). Thorough examination of primer 3 0 -OH-Me 2þ A alignment was first executed with nonhydrolyzable dNTP analogs with X family Pol b (Batra et al., 2008) and Pol k (Garcia- Diaz et al., 2007), revealing that the primer 3 0 -O coordinates the Me 2þ A as one of the ligands in the octahedral shell and exists 3.4 Å from the incoming nucleotide prepared for nucleophilic attack. However, exactly how and what docks the primer 3 0 -O into alignment and promotes its deprotonation and nucleophilic attack remained unclear.
Recent time-resolved x-ray crystallographic studies with X-family Pol b (Freudenthal et al., 2013;Kumar et al., 2022;Reed and Suo, 2017;Vyas et al., 2015), Pol k (Jamsen et al., 2022), and l (Jamsen et al., 2017), and Y-family Pol g (Nakamura et al., 2012) uncovered transient events that occur during correct nucleotide incorporation. By complexing Pol g with the correct, natural nucleotide dATP and Ca 2þ , Nakamura and co-workers showed that in the ground state without the Me 2þ A , the primer lies 4.1 Å away from the a-phosphate group with a bond angle of 169 , slightly misaligned for nucleophilic attack [Fig. 2(a)]. Correlated with the binding of Mg 2þ at the A-site, the 3 0 -OH of the primer was docked at 174 by the Mg 2þ A in the C2 0 -endo conformation to lie 3.3 Å from the a-phosphate. Coupled with product formation, the deoxyribose sugar ring at the primer end changes from a C2 0 -endo to C3 0 -endo sugar pucker conformation (Nakamura et al., 2012). Similarly, in X-family Pol b and Pol k, the primer 3 0 -O shifted over 1 Å closer to the a-phosphate upon Ca 2þ /Na 1þ to Mg 2þ /Mn 2þ exchange at the Me 2þ A binding site (Freudenthal et al., 2013;Jamsen et al., 2022;Kumar et al., 2022). These results highlight the contribution of Me 2þ A binding and its role on primer 3 0 -O alignment.

DEPROTONATION OF THE 3 0 -OH TERMINAL GROUP OF THE PRIMER
What exactly promotes primer deprotonation and whether it concertedly occurs with nucleophilic attack remain unclear and critical in understanding the catalytic mechanism of polymerases. During the in crystallo reaction with X-family Pol k, a water molecule lying close to the primer terminus was captured, suggesting its potential role to aid primer deprotonation (Jamsen et al., 2022). In contrast, in crystallo reaction by Pol b did not capture a water molecule interacting with the 3 0 -OH of the primer (Freudenthal et al., 2013). Instead, it was proposed that the amino acid side chain Asp256, which lies close to the primer, might be responsible for primer deprotonation. Computational studies indicate that Asp256 might facilitate primer deprotonation (Batra et al., 2013). However, it is important to note that this amino acid residue also coordinates the Me 2þ A , and mutating Asp256 will disrupt Me 2þ A binding and also indirectly affect deprotonation. In the study with Y-family Pol g, occurring simultaneously with Me 2þ A binding, a water molecule approached from behind and around the 2 0 carbon of the primer to possibly facilitate 3 0 -OH deprotonation (Nakamura et al., 2012). The protein residue Ser113, which lies close to the primer but does not coordinate the Me 2þ A , has also been suggested to promote primer deprotonation. In such close proximity to the primer terminus, the water molecule or Ser113 can easily accept the primer 3 0 -O proton. However, simultaneously displacing the water molecule and mutating amino acid residue 113 from serine to an alanine did not reduce catalytic efficiency (Gregory et al., 2021). Therefore, primer deprotonation in Pol g was proposed to most likely occur through multiple pathways, and a well-defined general base is not needed.

PRIMER TERMINUS ALIGNMENT DURING INCORRECT SUBSTRATE DISCRIMINATION
During the incorporation of the incorrect nucleotide, the role of the Me 2þ A and the importance of primer terminus alignment were further pronounced. In static crystal structures of Pol g with the incorrect nucleotide at the insertion site, the primer is positioned misaligned in the up-shifted conformation, restricted from attacking the aphosphate (Zhao et al., 2013). A water molecule coordinates the Me 2þ A in place of the primer 3 0 -O. Notably, the degree of primer misalignment varies depending on the DNA sequence near the primer terminus, which corresponds to the different misincorporation efficiencies reflected in the kinetic assays (Zhao et al., 2013). During the in crystallo reaction with Pol g, the majority of the primer terminus exists in the misaligned up-shifted conformation near the roof of the active site in the absence of Me 2þ A (Chang et al., 2022). Upon Mg 2þ binding to the Me 2þ A binding site, the primer terminus is docked in the aligned conformation [ Fig. 2(b)]. Nevertheless, the primer terminus exists 2.5 Å away from the Me 2þ A , still 0.5 Å longer than the optimal distance for Mg 2þ coordination, suggesting major barriers in primer deprotonation and alignment. Similarly, during the in crystallo reaction with Pol b incorporating a dATP across a dG template, the primer terminus showed weak electron densities (r less than 3.0 r.m.s.d for the F o -F c omit map), suggesting it is partially destabilized (Freudenthal et al., 2013). The suboptimal coordination distance of the Me 2þ A to the primer end and this misaligned conformation in both polymerase systems, which places the primer terminus 3 0 -O in an unfavorable position for nucleophilic attack, explain the significant decrease in catalytic activity during misincorporation. Furthermore, it was previously established that Mn 2þ can promote error-prone synthesis of polymerases. Titrating the polymerases in crystallo revealed that Mn 2þ was more optimal than Mg 2þ at aligning the primer end in Pol g, and binding of Mn 2þ linearly correlates with primer alignment even during incorrect nucleotide incorporation (Chang et al., 2022). Such mutagenic property of Mn 2þ was also previously hinted by static crystal structures of Pol k. The structures of Pol k incorporating dCTP against a dG revealed that Mn 2þ promotes the sugar pucker to exist in the C3 0 -endo conformation as opposed to the C2 0 -endo conformation, aiding the primer 3 0 -O to exist even closer to the target a-phosphate (Garcia-Diaz et al., 2007). These results further highlight a correlation between the Me 2þ A , primer alignment, and polymerase fidelity. Rearrangements at the primer terminus and the Me 2þ A coordination environment in the polymerase active site have been observed during DNA damage bypass and nucleotide analog drug insertion. Phenanthriplatin is a potential cisplatin-analog cancer drug that covalently links to DNA and obstructs DNA synthesis and transcription Kellinger et al., 2013;Park et al., 2012). Similarly, cyclopurines like 8,5 0 -cyclo-2 0 -deoxyadenosine also stall the DNA replication machinery (Kuraoka et al., 2001). When Pol g contacts 8,5 0 -cyclo-2 0 -deoxyadenosine or phenanthriplatin on the DNA template, the primer was found to misalign, preventing nucleotide incorporation (Gregory et al., 2014;Weng et al., 2018). The primer end aligned with the incoming nucleotide, and bypass occurred only in the presence of mutagenic Mn 2þ . Similarly, the drug (-)-b-L-2 0 ,3 0dideoxy-3 0 -thiacytidine (lamivudine) binding at the Pol b insertion site resulted in primer terminus misalignment and perturbation of the Me 2þ A octahedral coordination geometry. These structural results were reflected in lamivudine's tighter binding affinity and 323-fold less efficient incorporation rate than the natural substrate dCTP (Vyas et al., 2017). In Pol b, Arg283 stabilizes the syn-conformation of 8oxo-2 0 -deoxyguanosine (8-oxodG) through hydrogen bonding on the template. Such interaction shifts 8-oxodG into a geometry that favors base-pair binding of dATP, allowing dCTP to be incorporated over FIG. 2. Primer terminus alignment during DNA synthesis. (a) Primer terminus alignment during correct incoming nucleotide incorporation prior to nucleophilic attack. Superimposition of the ternary structure of Pol g after 40 s 1.0 mM Mg 2þ soaking (4ECR) in gray and the primer terminus with no soaking (4ECQ) in light gray during dATP incorporation across a dT indicates movement in the primer prior to nucleophilic attack. b, Primer terminus alignment during incorrect incoming nucleotide incorporation prior to nucleophilic attack. Superimposition of the ternary structure of Pol g after 40 s 1.0 mM Mg 2þ soaking (7U77) in gray and the primer terminus with no soaking (7U72) in light gray during dGTP misincorporation across a dT indicates that the primer is dynamic prior to nucleophilic attack. The Mg 2þ A and Mg 2þ B are represented with solid green spheres, while the Mg 2þ C binding site is represented with a dotted unfilled circle. (c) Proposed kinetic model during DNA polymerase correct nucleotide incorporation (red pink) and misincorporation (blue). During correct nucleotide incorporation, the primer 3 0 -OH becomes well-aligned by the Me 2þ A , and the reaction proceeds right after Me 2þ C binding. Catalysis occurs in conjunction to Me 2þ C binding and Me 2þ A dissociation.
Structural Dynamics ARTICLE pubs.aip.org/aip/sdy dATP by only 2-folds. When Arg283 is mutated to Lys in Pol b, 8-oxodG existed in the anti-conformation and shifted the template upstream by 2 Å , tugging the primer away from the Me 2þ A to exist 139 out of line with the a-phosphate unable to attack the incorrect nucleotide dATP (Freudenthal et al., 2012). Thus, primer alignment might be an innate mechanism that safeguards against the bypass or incorporation of some damaged substrates.
Furthermore, chemical modifications on the sugar moiety of nucleotides can affect the dynamics of primer alignment and incorporation efficiency. When a ribonucleotide (rN), which has one additional oxygen atom on C2 0 compared to a deoxynucleotide (dN), existed at the primer terminus, the primer terminus was found to be in the aligned conformation even in the absence of the Me 2þ A during correct nucleotide incorporation of Pol g (Gregory et al., 2021). Recent studies show that misincorporation is favored during Pol gmediated extension from a rN (Chang et al., 2023). Crystal structures with rN at the 3 0 -end of the DNA duplex showed improvement in primer alignment even when the incorrect dGTP nucleotide was bound at the insertion site across from a template dT. Not only was the primer terminus more favored in the docked position for nucleophilic attack, but also the sugar motifs of these rN existed in the C3 0endo sugar pucker conformation, which is the favored conformation in the product state, as the 3 0 -O of the primer lies closer to the target a-phosphate (Batra et al., 2006;Berman et al., 2007;Doubli e et al., 1998;Franklin et al., 2001;Garcia-Diaz et al., 2007;Li et al., 1998;Nakamura et al., 2012;Wang and Yang, 2009). Cytarabine (araC) is a dC mimic that contains a -OH group in the b direction on the 2 0 carbon of the arabinose sugar moiety. Static structures show that at the primer terminus, araC exists in both aligned and misaligned conformations even during correct nucleotide incorporation. Moreover, this dC mimic drug exists in the C2 0 -endo conformation and misaligned with the a-phosphate, explaining how araC inhibits DNA extension even though it contains a functional 3 0 -OH group (Rechkoblit et al., 2018). These results again showcase the importance of primer alignment and suggest small changes on the sugar ring can significantly impact incorporation efficiency.

TRANSIENT METAL ION BINDING
Based on static crystal structures, Steitz proposed that two-metal ions are required and sufficient for polymerase catalysis (Steitz and Steitz, 1993). The Me 2þ B stabilizes the incoming nucleotide by coordinating with its phosphate oxygens; the Me 2þ A helps to decrease the pKa of the primer for deprotonation and nucleophilic attack. Recent in crystallo reaction studies revealed an additional divalent metal ion, or the C site metal ion (Me 2þ C ) binds between the Pa and Pb of the incoming nucleotide during reaction (Freudenthal et al., 2013;Nakamura et al., 2012;Vyas et al., 2015;Yang et al., 2016). Instead of protein sidechains, it is coordinated by four water molecules and two oxygens on the newly formed DNA strand and pyrophosphate. It has only been captured during catalysis but not detected in static structures with various inhibitors. Thus, the Me 2þ C was not detected in decades of static structures of polymerases and remains hard to probe. The exact timing of Me 2þ C binding and its role in DNA synthesis remain controversial. It has been proposed that the Me 2þ C stabilizes the product state, pushes the reaction forward, inhibits the forward reaction, induces the reverse reaction, and/or helps with damage bypass.
Initially, the Me 2þ C has been suggested to bind to the newly formed DNA and pyrophosphate to stabilize the product state. In Pol g, the Me 2þ C was captured simultaneously with production formation, except at the very beginning of the reaction (Nakamura et al., 2012). In Pol b (Freudenthal et al., 2013) and l (Jamsen et al., 2017), during correct nucleotide incorporation, the electron density of the Me 2þ C appears slightly right after the detection of new bond density. Concurrently or immediately after, the Me 2þ A dissociates from the active site. Thus, the role of the Me 2þ C has been suggested to help reduce the electronegative buildup of the newly formed DNA backbone and also to help induce pyrophosphate protonation and release [ Fig. 3(b)].
Moreover, the Me 2þ C potentially drives the reverse reaction of phosphorolysis. The Me 2þ C coordinates the newly formed DNA and pyrophosphate oxygens at 2.0-2.2 Å . Just like how the Me 2þ A closely coordinates the primer terminus and helps to deprotonate the primer 3 0 -OH, the Me 2þ C is optimally positioned to deprotonate one of pyrophosphate's oxygen. Once deprotonated, the more electronegative charged oxygen can undergo nucleophilic attack on the newly formed product phosphate to break the newly formed bond. Hence, it has been suggested that the role of the Me 2þ C lies in deprotonating pyrophosphate (Freudenthal et al., 2013). Nevertheless, phosphorolysis has never been visualized with time-resolved crystallography before, as the The reactant state is colored in yellow, while the product state is colored in blue. The red spheres represent the water molecules that coordinate the octahedral shell of the Mg 2þ C that is colored in green. (b) Proposed free energy diagram for the two-metal ion dependent (green) and three-metal ion dependent (yellow or blue) DNA synthesis. The Mg 2þ C may bind prior to catalysis and drive the forward reaction (yellow) or bind after catalysis to stabilize the converted product (blue).

Structural Dynamics
ARTICLE pubs.aip.org/aip/sdy forward reaction is far more efficient than the reverse reaction. However, quantum mechanical/molecular mechanical simulations show that the Me 2þ C can reduce the energy barrier for phosphorolysis to occur .
Recent evidence suggests that the Me 2þ C may be required to drive the DNA synthesis reaction. With Mn 2þ , which is more electron dense and, thus, can be detected at low occupancies, it was shown that Me 2þ C binding perfectly correlates with production formation even during early stages of the reaction in Pol g . Recent studies with cross-linked Pol b also showed the Me 2þ C binding simultaneously with phosphodiester formation (Kumar et al., 2022). Moreover, extensive in crystallo titration with Pol g found that the A and B sites are high affinity binding sites (<0.5 mM), while the C site is a weak binding site with a low binding affinity of $3 mM, similar to what has been found in solution. When only the A site and B sites are saturated, the primer 3 0 -O, a-phosphate, and bridging oxygen colinearize and are inclined for nucleophilic attack, but the reaction product is not observed even after long reaction time. Only after Me 2þ C binding, product formation did occur, suggesting the essential role of the Me 2þ C during catalysis. Furthermore, when a sulfur element, which is less electronegative than oxygen, replaces the incoming nucleotide's Sp oxygen, which serves as a coordinating ligand to the Me 2þ C , density surrounding the usual Me 2þ C is not captured, and the Me 2þ binding affinity is reduced by 25-fold from the kinetic assays . During slow mismatch incorporation, the transient Me 2þ C was captured binding before product formation (Chang et al., 2022). Initially, it binds between the Pa and Pb oxygens but exists slightly too far from the Pa oxygen of the incoming nucleotide. After the Me 2þ C enters the active site in between the Pa and Pb oxygens and shifts downward to form an optimal octahedral geometry, the a-b-phosphate bond breaks. Thus, the in crystallo studies with Pol g suggested that Me 2þ C binding proceeds catalysis, and that the Me 2þ C may be directly involved in reducing the energy barrier for ab-phosphate bond breakage [ Fig. 3(b)].
This proposed catalytic role of the Me 2þ C has been challenged in recent years. Wang and colleagues point out that because the occupancy of the Me 2þ C is always lower than that of the product pyrophosphate, the Me 2þ C must bind after reaction in the product complex and, thus, cannot actively push the catalytic reaction forward (Wang and Smithline, 2019). However, it has been argued that time-resolved experiments performed by hand cannot differentiate whether the Me 2þ C binds before or after reaction, as Me 2þ C binding can occur on the order of sub-seconds (Tsai, 2019). They also argued that the loss of the Me 2þ C binding after product DNA and pyrophosphate formation could explain why the occupancy of the Me 2þ C is always lower than that of the product pyrophosphate. While computational studies argue that Mg 2þ or Mn 2þ cannot favorably bind between the Pa and Pb oxygens of the incoming nucleotide due to suboptimal bond lengths, geometry angle, and electrostatic charges of the oxygens, Chang and colleagues captured Mg 2þ and Mn 2þ binding near the Me 2þ C binding site prior to reaction (Chang et al., 2022). Furthermore, high concentrations of Mg 2þ or Mn 2þ used in in crystallo soaking experiments have been shown to inhibit the polymerase reaction in solution (Frank and Woodgate, 2007;Wang and Konigsberg, 2022). Thus, the Me 2þ C has been suggested to remain bound with the product pyrophosphate within the active site. At this location, it can induce the reverse reaction and hold the product pyrophosphate within the active site. With the substrate unable to enter, additional DNA synthesis cannot occur at high metal ion concentrations.
It has also been proposed that transient divalent metals can help during the incorporation of damaged substrates. During the incorporation of the anti-form of 8-oxo-7,8-dihydro-2 0 -deoxyguanosine triphosphate (8-oxoGTP), which is sterically destabilized within the nucleotide binding pocket in Pol b, minimal distortion of the active site was observed. Instead, a divalent metal ion was captured binding near the Pa and Pb oxygens prior to reaction to possibly alter the electrostatic nature of the a-phosphate oxygens to favor nucleophilic attack . However, another study on dCTP incorporation across an (anti) 8-oxoGTP found the Me 2þ C only binds during the catalytic reaction (Vyas et al., 2015). Similarly in X-family Pol l, after crystals were soaked in 50 mM Mn 2þ , density close to the Me 2þ C was captured and found coordinating the C8 0 oxygen of 8-oxodGTP and a nearby aspartate indirectly through a water molecule (Jamsen et al., 2021). Furthermore, an additional divalent metal site was captured near the Me 2þ A binding site in Pol g during the incorporation of Sp-dATPaS, which has the Sp oxygen replaced with a sulfur element . While it remains unclear if these densities found in Pol b, l, and g truly represent the Me 2þ C , it is likely that one additional or more transiently bound divalent metal ions play the role of stabilizing strained substrates for more efficient incorporation.
In addition to DNA polymerases, it has also been proposed that all RNA polymerases (Steitz, 1998), many nucleases (Stahley and Strobel, 2005;Toor et al., 2009;Toor et al., 2008), and ribozymes (Steitz and Steitz, 1993) follow the two-metal-ion dependent catalysis mechanism. Similar time-resolved crystallography experiments revealed an additional divalent Me 2þ binding transiently during RNA cleavage in RNaseH (Samara and Yang, 2018). Moreover, the authors further discovered two additional monovalent Me 1þ binding near the RNA backbone, possibly facilitating the water-mediated deprotonation and nucleophilic attack as well as stabilizing the transition state. The existence of the third Me 2þ has also been suggested by studies on Endonuclease V, which was proposed to use a two-metal-ion dependent mechanism for cutting RNA. Similar as in RNaseH, a third divalent Me 2þ binds transiently to direct the nucleophilic water molecule during Endonuclease V catalysis (Wu et al., 2019). Furthermore, a transient Me 2þ has also been sighted in MutT, which initially was proposed to catalyze 8-oxodGTP hydrolysis with two divalent Me 2þ (Nakamura and Yamagata, 2022). The intermediate structures showed an additional metal ion binding in between two protein residues and directing a nucleophilic water closer and aligned to the scissile phosphate. In each of these three systems, the transient divalent Me 2þ binds on the side of the nucleophilic water molecule, aligning it for nucleophilic attack. It is possible that many more metalloenzymes use transient metal ions during catalysis to stabilize substrate, product, and/or promote transition state formation.

CONCLUSION
Time-resolved crystallography has extensively increased the understanding of DNA polymerases by revealing transient events like primer alignment, role of the Me 2þ A , and the presence of transient metal ions that occur during nucleotidyl transfer and DNA repair. However, in crystallo visualization of DNA synthesis remains limited to only X-and Y-family polymerases, which are specialized in DNA repair and damage bypass, possibly because polymerases that function during DNA replication such as A-and B-family polymerases perform catalysis too rapidly to be observed in crystallo. Furthermore, polymerases that exhibit large global conformation changes, diffract to subatomic resolutions lower than 3 Å , or are intolerant of cofactor soaking would present challenges for in crystallo studies. In addition, this manual metal ion diffusion technique is limited by how fast the crystallographer can feasibly transfer ternary complex crystals between buffer solutions, which is typically limited to 10 s. Automated stop flow equipment coupled with x-ray free electron lasers may further improve the temporal resolution of transient observations and lead to discoveries of new protein dynamic events before or during catalysis (Olmos et al., 2018). Nevertheless, the number of diverse enzymes being studied with time-resolved crystallography continues to increase, highlighting the essential roles of active site dynamics and transient element binding during enzyme catalysis.